Gene probes are important in the medical diagnostic area. The identification of genetic material in an organism can help to predict the predisposition of that organism to certain diseases. In addition, the identification of genetic material from infectious organisms can help to pinpoint the causation of diseases.
The gene probe area has been developing for several years, and its technical and popular literature has been prolific. See, for example, Klausner et al, Bio/Technology, Aug., 1983, p. 471; Bylinsky, Fortune, Jul. 9, 1984, p. 140; Engleberg, ASM News, Vol. 57, No. 4, 1991, p. 183; Gillespie, Veterinary Microbiology, 24 (1990) 217.
In gene probe assays, one of the key reagents utilized is the nucleic acid probe conjugated to an active molecular species. A nucleic acid probe is a single-stranded nucleic acid sequence that will combine (anneal) with a complementary single-stranded target nucleic acid sequence to form a double-stranded molecule (hybrid). The probe can either be totally synthetic, or from a biological source (recombinant DNA), or some combination of the two.
The active molecular species (also referred to as labels) can include such things as hapten, ligand or luminescent label. A hapten is an incomplete antigen, incapable by itself to provoke an immune response but, when suitably attached to another molecule, becomes capable of producing antibodies which will specifically recognize the hapten. A ligand is any compound for which a receptor naturally exists or can be prepared. A receptor is any compound capable of recognizing a particular spatial and polar organization of a molecule, i.e., epitopic site. Illustrative receptors include antibodies, enzymes, antibody fragments (such as Fab fragments), lectins, complement components, rheumatoid factors, hormones, avidin, staphylococcal protein A, and the like. Luminescent labels include such things as organic chemicals, organic metal chelate complexes or biopolymers capable of emitting light of longer wavelengths upon being excited with various sources of energy, introduced either in the form of light of shorter wavelengths (fluorescence) or derived internally from the molecule itself as a result of chemical conversion (chemiluminescence).
In some assays, for example in a sandwich-formatted assay, two kinds of nucleic acid probe conjugates (capturing probe and signalling probe) can be utilized. The main function of hapten or ligand in the capturing probe is to mediate the capturing of hybridized (annealed) nucleic acids (consisting of the target nucleic acid and the capture probe) by complexing with the anti-hapten IgG or the receptor immobilized on a solid phase. Through hybridizing to the neighboring region of the target nucleic acid, signalling probe facilitates the detection of the sandwich hybrid by virtue of its incorporated signal label that either is capable of signal producing or can be specifically bound to a binding protein which in turn carries or is capable of producing amplified signals. An amplified signal can be exemplified by a biotin/avidin system. Avidin, being a large molecule, can be reacted with several acridinium ester groups, so that the resulting modified avidin molecule has a much greater signal than if only one acridinium ester was present. Thus an "amplified" signal is obtained. A further example of a binding protein carrying an amplified signal is the receptor/liposome system. Liposome, being a cell-mimicing vescicle, can encapsulate thousands of suitably modified acridinium esters (hydrophilic acridinium esters) or other luminescent compounds and be subsequently attached to a number of receptors on its outer surface by covalent linkages. (See copending patent application Ser. No. 07/226,639, filed Aug. 1, 1998, abandoned in favor of file-wrapper-continuation application Ser. No. 07/826,186, filed Jan. 22, 1992, now U.S. Pat. No. 5,227,489, issued Jul. 13, 1993.) Examples of both capturing and signal probes are shown in FIGS. 1 and 2.
The type of signal is not critical for our purposes and can include radioactive, luminescent, phosphorescent, and other types of signals, for example the use of acridinium esters, phycobiliproteins, and the indirect biotin-avidin, or alkaline phosphatase systems.
Many techniques for coupling the probes with active molecular species are known. These include (1) modifying the purine or pyrimidine moiety of the nucleotide or (2) attaching certain functional groups to the 5'- or 3'- ends of the probe. Even though the latter approach of functionalizing the 5'- or 3'- ends of nucleic acids is generally preferred because interference between the probes and target nucleic acids can be minimized, the techniques herein can also be utilized to modify the individual prederivatized bases of the polynucleotides.
Synthesis of polynucleotides of even more than 100 bases in length has become common practice since the commercialization of DNA synthesizers, which employ, among other techniques, the phosphoramidite coupling chemistry. Several techniques of functionalizing probes at the 5'- or 3'- ends have been reported. Agrawal et al [Nucleic Acid Res., 14 (1986) 6227] attached a ribonucleoside to the 5'- end of the deoxyoligonucleotide, oxidizing the ribosyl moiety with periodate to generate the functional groups of dialdehyde for coupling. Furthermore, a series of deoxyoligonucleotide modifiers containing homologous aminoalkyl or mercaptoalkyl groups have been made commercially available (e.g., by Clontech Laboratories, Inc., Palo Alto, Calif.). These can be added wherever desired (3'- end, middle of the oligomer chain, or 5'- end) during preparing the oligonucleotides on an automated synthesizer.
When an aminoalkyl-functionalized probe is selected for coupling, ligands carrying a variety of functional groups can be utilized to form stable conjugates. For example, the use of fluorescein with isothiocyanate (FITC), an already activated form of ligand, has been reported in Nucleic Acid Res., Vol. 13 (1985) 1529. The use of biotin with carboxylate, reported in Nucleic Acid Res., Vol. 14 (1986) 6227, requires preactivation with N-hydroxysuccinimide (NHS) to form biotin-NHS. However, in both cases, vast excess of ligands, exceeding a molar ratio of 500:1, were needed to drive the reaction and to maximize the usage of synthetic oligonucleotide. The adoption of such a high loading ratio has been widespread among those who practice this coupling chemistry and is believed to be necessary because of the aqueous lability and/or mild reactivity of these two activation forms. The yield of probe conjugate from these reactions is reasonably high (greater than 80%). However, the disadvantages are those associated with use of large excesses of one reactant, namely tedious work-up, difficult purification, and cost.